Sphingosine pathway modulating compounds for the treatment of cancers

ABSTRACT

The invention provides methods and compositions for treating cancers and myeloproliferative disorders using sphingosine kinase-1 inhibitors, such as SK1-I, and selective sphingosine-1-phosphate receptor agonists, such as ozanimod.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 62/524,221 filed Jun. 23, 2017 which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical treatment ofcancers.

BACKGROUND

Sphingosine-1-phosphate (S1P) was discovered to be a bioactive signalingmolecule over 20 years ago. Studies have since identified two relatedkinases, sphingosine kinase 1 and 2 (a/k/a sphingosine kinase “type I”and “type II” respectively, and SphK1 and SphK2 respectively), whichcatalyze the phosphorylation of sphingosine to S1P. Extracellular S1Pcan bind to and activate each of five S1P-specific, G protein-coupledreceptors (designated S1PR₁₋₅) to regulate cellular and physiologicalprocesses in an autocrine or paracrine manner. Selective inhibitors ofeach of sphingosine kinase 1 and 2, as well as both non-selective andselective agonists of S1PRs, have been developed and are known in theart.

SUMMARY

One embodiment of the invention provides a method for treating livercancer, such as hepatocellular carcinoma (HCC), in a mammalian subject,such as a human, that includes the step of:

-   -   administering to a mammalian subject in need of treatment for        liver cancer, a therapeutically effective amount of a        sphingosine kinase type I inhibitor, such as SK1-I or a        pharmaceutically acceptable salt thereof.

A related embodiment of the invention provides a pharmaceuticalcomposition that includes a sphingosine kinase type I inhibitor, such asSK1-I or a pharmaceutically acceptable salt thereof for the treatment ofliver cancer, such as HCC, in a mammal, such as a human patient.

Another embodiment of the invention provides a method for treating acancer or a myeloproliferative disorder (myeloproliferative neoplasm) ina mammalian subject, such as a human, that includes the step of:

-   -   administering to a mammalian subject in need of treatment for a        cancer or myeloproliferative disorder, a therapeutically        effective amount of a sphingosine-1-phosphate receptor agonist,        such as an agonist of one or both of sphingosine-1-phosphate        receptor-1 (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅)        such as ozanimod (RPC1063) or a pharmaceutically acceptable salt        thereof.

A related embodiment of the invention provides a pharmaceuticalcomposition for the treatment of a cancer or a myeloproliferativedisorder (myeloproliferative neoplasm) in a mammalian subject, such as ahuman, that includes:

-   -   a therapeutically effective amount of a sphingosine-1-phosphate        receptor agonist, such as an agonist of one or both of        sphingosine-1-phosphate receptor-1 (S1P₁) and        sphingosine-1-phosphate receptor-5 (S1P₅) such as ozanimod        (RPC1063) or a pharmaceutically acceptable salt thereof.

Still another embodiment of the invention provides a method for treatinga cancer or a myleoproliferative disorder (myeloproliferative neoplasm),such as any of those disclosed herein, in a mammalian subject, such as ahuman, including the step of:

-   -   co-administering to a mammalian subject in need of treatment for        a cancer or myeloproliferative disorder, a therapeutically        effective amount of:        -   (a) a sphingosine kinase type I inhibitor, such as one            disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, such            as SK1-I, or a pharmaceutically acceptable salt thereof; and        -   (b) one or more immune checkpoint inhibitors, which may be            monoclonal antibodies, such as one or more selected from the            group consisting of: PD-1 inhibitors such as mAbs            Pembrolizumab (Keytruda®) and Nivolumab (Opdivo®); PD-L1            inhibitors such as mAbs Atezolizumab (Tecentriq®), Avelumab            (Bavencio®), and Durvalumab (Imfinzi®); and CTLA-4            inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain Ig            Suppressor of T Cell Activation (VISTA) inhibitors such as            mAb JNJ-61610588 (ImmuNext Inc.).

Additional features, advantages, and embodiments of the invention may beset forth or apparent from consideration of the following detaileddescription, drawings if any, and claims. Moreover, it is to beunderstood that both the foregoing summary of the invention and thefollowing detailed description are exemplary and intended to providefurther explanation without limiting the scope of the invention asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows MTT assay data (72 hours) for various concentrations ofozanimod for four human hepatocellular carcinoma cell lines.

FIG. 2 shows MTT assay data (72 hours) for various concentrations ofABC294640 for four human hepatocellular carcinoma cell lines.

FIG. 3 shows MTT assay data (72 hours) for various concentrations ofSK1-I for four human hepatocellular carcinoma cell lines.

FIG. 4A shows MTT assay data (48 hours) for various concentrations ofSK1-I for three human hepatocellular carcinoma cell lines. FIG. 4B showsMTT assay data (48 hours) for various concentrations of PF-543, a superpotent SphK1 inhibitor, for the same three human hepatocellularcarcinoma cell lines shown in FIG. 4A.

FIGS. 5A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Huh7 cells. SK1-I strongly induced apoptosis inthe Huh7 cells.

FIGS. 6A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for PLC-PRF5 cells. SK1-I strongly induced apoptosisin the PLC-PRF5 cells.

FIGS. 7A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Hep 3B cells. SK1-I strongly induced apoptosisin the Hep 3B cells.

FIG. 8A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Hep G2 cells. SK1-I strongly induced apoptosisin the Hep G2 cells.

FIGS. 9A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Huh7 cells. Ozanimod strongly inducedapoptosis in the Huh7 cells.

FIGS. 10A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for PLC-PRF5 cells. Ozanimod stronglyinduced apoptosis in the PLC-PRF5 cells.

FIGS. 11A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep 3B cells. Ozanimod strongly inducedapoptosis in the Hep 3B cells.

FIGS. 12A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep G2 cells. Ozanimod strongly inducedapoptosis in the Hep G2 cells.

FIGS. 13A-D shows apoptosis assay data for various concentrations ofABC294640 and no-drug control for Huh7 cells. ABC294640 failed to induceapoptosis in the Huh7 cells.

FIGS. 14A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for PLC-PRF5 cells. ABC294640 did notsubstantially induce apoptosis in the PLC-PRF5 cells.

FIGS. 15A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep 3B cells. ABC294640 did notsubstantially induce apoptosis in the Hep 3B cells.

FIGS. 16A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep G2 cells. ABC294640 failed toinduce apoptosis in the Hep G2 cells.

FIGS. 17A-C show apoptosis assay data for various concentrations ofSK1-I and no-drug control for Jurkat cells (human T-cell leukemia cellline). SK1-I strongly induced apoptosis in the Jurkat cells.

FIGS. 18A-C show apoptosis assay data for various concentrations ofozanimod and no-drug control for Jurkat cells. Ozanimod strongly inducedapoptosis in the Jurkat cells.

FIGS. 19A-D show apoptosis assay data for various concentrations ofSK1-I and no-drug control for primary human hepatocytes. SK1-I did notinduce apoptosis of the primary human hepatocytes at any concentrationtested.

FIGS. 20A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for primary human hepatocytes. Ozanimod didnot induce apoptosis of the primary human hepatocytes at anyconcentration tested.

DETAILED DESCRIPTION

The invention provides new uses of sphingosine kinase-1 inhibitors, suchas SK1-I, and selective sphingosine-1-phosphate receptor agonists, suchas ozanimod, for treating cancers, such as a liver cancer, andmyeloproliferative neoplasms (myeloproliferative disorders), in mammals,such as human patients. The invention also provides new uses ofselective sphingosine kinase type I inhibitors, such as SK1-I, andselective sphingosine-1-phosphate receptor agonists, such as ozanimod,for inducing apoptosis and/or necrosis of mammalian, such as human,cancer cells, such as liver cancer cells, and myeloproliferativeneoplasm cells.

Sphingosine kinase 1 inhibitors used in various embodiments of theinvention may, for example, include any of those disclosed in U.S. Pat.Nos. 8,372,888 and/or 8,314,151, each of which is hereby incorporated byreference in its entirety herein, or pharmaceutically acceptable saltsthereof. The sphingosine kinase I inhibitor may, for example, be (E,2R,3S)-2-(methyl amino)-5-(4-pentylphenyl)pent-4-ene-1,3-diol (also known asSK1-I), or a pharmaceutically acceptable salt thereof such as but notlimited to a hydrochloride salt. The structure of SK1-I is shown below.

See also Paugh et al., Blood, 2008 112: 1382-1391.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

or a pharmaceutically acceptable salt of the compound such as but notlimited to a hydrochloride salt.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

wherein R is selected from a straight carbon chain, a branched carbonchain, a straight carbon chain comprising one or more heteroatoms, abranched carbon chain comprising one or more heteroatoms, a cyclic ring,a heterocyclic ring, an aromatic ring, a hetero-aromatic ring, or anycombination of the foregoing,

or a pharmaceutically acceptable salt thereof such as but not limited toa hydrochloride salt.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

wherein R is 3,4-dimethoxy, 4-phenyl or 3-pentyl,

or a pharmaceutically acceptable salt thereof such as but not limited toa hydrochloride salt.

Sphingosine-1-phosphate receptor agonists used in various embodiments ofthe invention may, for example, be any of those disclosed in any of U.S.Pub. Nos. 20110172202, 20130231326, and 20150299149, or pharmaceuticallyacceptable salts thereof. The agonists may be agonists of one or both ofsphingosine-1-phosphate receptor-1 (S1P₁) and sphingosine-1-phosphatereceptor-5 (S1P₅) and may have little or at least no substantial agonistactivity against other sphingosine-1-phosphate receptors (in a mammalsuch as a human). The sphingosine-1-phosphate receptor agonist used may,for example, be5-[3-[(1S)-1-(2-hydroxyethylamino)-2,3-dihydro-1H-inden-4-yl]-1,2,4-oxadiazol-5-yl]-2-propan-2-yloxybenzonitrile(also known as ozanimod and RPC1063) or a pharmaceutically acceptablesalt thereof such as but not limited to a hydrochloride salt. Thestructure of ozanimod is shown below.

See also Scott et al., British Journal of Pharmacology 2016173:1778-1792.

The sphingosine-1-phosphate receptor agonist may, for example, beetrasimod or a pharmaceutically acceptable salt thereof such as but notlimited to a hydrochloride salt. The structure of etrasimod is shownbelow.

The sphingosine-1-phosphate receptor agonist may, for example, beamiselimod or a pharmaceutically acceptable salt thereof such as but notlimited to a hydrochloride salt. The structure of amiselimod is shownbelow.

ABC294640 (also known as Yeliva®) used in the experiments presentedherein is a reported sphingosine kinase-2 selective inhibitor, namelythe compound (7S)-3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)adamantane-1-carboxamide.The structure of ABC294640 is shown below.

See also French et al., J. Pharmacol. Exp. Ther. 2010, 333, 129-139.

Experiments

MTT cell viability assays evaluating the effect of differentconcentrations of each of ozanimod, ABC294640 and SK1-I on four humanhepatocellular carcinoma cell lines, Hep G2, Hep 3B, Huh 7 and PLC-PRF5were performed. These four cells line were selected for the studybecause they are among HCC cells lines whose gene expression profilesmost closely resemble those of primary HCC tumors. See Chen et al., BMCMedical Genomics 2015, 8(Suppl 2):S5. The following concentrations ofthe compounds were tested.

Ozanimod: 200 μM, 111.1 μM, 61.73 μM, 34.29 μM, 19.05 μM, 10.58 μM, and0 μM. ABC294640: 200 μM, 111.1 μM, 61.73 μM, 34.29 μM, 19.05 μM, 10.58μM and 0 μM. SK1-I: 20 μM, 11.11 μM, 6.173 μM, 3.429 μM, 1.905 μM, 1.058μM and 0 μM.

The following MTT assay protocol was followed.

-   -   Prepared stock solutions: 50 mM ozanimod, 50 mM ABC294640, 10 mM        SK1-I in DMSO.    -   Plated 20000 cells in 160 μl medium per well in 96-well plates        for each cell line and incubated at 37° C. overnight.    -   Prepared compound serial dilutions: for ozanimod and ABC294640:        dilute stock 50 mM 1:50 in medium to 1000 μM; for SK1-I, dilute        stock 10 mM 1:50 in medium to 100 μM, them make serial 1:1.8        fold serial dilution to the titration. For negative control,        diluted DMSO 1:50 to medium.    -   Added 40 μl negative control and serially titrated compounds to        160 μl of cells for each cell line. Performed in triplicate for        each cell line.    -   Incubated at 37° C. for 72 hours.    -   Performed assay using Vybrant MTT Cell Proliferation Assay kit        (V-13154) (Molecular Probes) from Thermo Fisher Scientific        (Waltham, Mass. USA).        The results of these 72-hour treatment MTT assays are shown in        FIGS. 1-3 as follows.

FIG. 1 shows the MTT assay data (72 hours) for ozanimod for the fourhuman hepatocellular carcinoma cell lines.

FIG. 2 shows the MTT assay data (72 hours) for ABC294640 for the fourhuman hepatocellular carcinoma cell lines.

FIG. 3 shows the MTT assay data (72 hours) for SK1-I for the four humanhepatocellular carcinoma cell lines.

48-hour treatment MTT assays were also performed as follows. FIG. 4Ashows MTT assay data (48 hours) for various concentrations of SK1-I forthree human hepatocellular carcinoma cell lines, Hep G2, Huh 7, andPLC-PRF5. FIG. 4B shows MTT assay data (48 hours) for variousconcentrations of PF-543, a super potent SphK1 inhibitor (see Schnute etal., Biochem. J. (2012) 444, 79-88), for the same three humanhepatocellular carcinoma cell lines shown in FIG. 4A. This data showsthat SK1-I is more effective at killing hepatocellular carcinoma cellsthan PF-543 despite the latter drug's much greater potency in inhibitingSphK1.

Apoptosis/necrosis assays evaluating the effect of differentconcentrations of each of ozanimod, ABC294640 and SK1-I on the fourhuman hepatocellular carcinoma cell lines, Hep G2, Hep 3B, Huh 7 andPLC-PRF5 were also performed.

The protocol used for the apoptosis assays was:

-   -   Plated cells in 6-well plates and incubated at 37° C. overnight.    -   Prepared concentrations of test compound and control compound        DMSO in media.        -   Ozanimod: 5 μM, 10 μM, and 20 μM.        -   ABC294640: 10 μM, 20 μM, and 40 μM.        -   SK1-I: 10 μM, 20 μM, and 40 μM.        -   Control: 0.2% DMSO.    -   Aspirated the medium from the 6-well plates and added test        compound concentrations in media or 0.2% DMSO control in media.        Incubated at 37° C. for 24 hours.    -   Collected and processed the cells following the flow cytometry        protocol of the GFP Certified® Apoptosis/Necrosis detection kit        from Enzo Life Sciences, Inc. (product no. ENZ-51002;        Farmingdale, N.Y., USA).

FIGS. 5-16 present graphs plotting the data from these 24-hour apoptosisassays for the different concentrations of compounds and no-drug controlfor the various cell lines. Channel FL2 picks up the apoptosis signaland channel FL3 picks up the necrosis signal. Data points in Quadrant 3(Q3) in the graphs corresponds to cells undergoing apoptosis (cellspositive for the apoptosis detection reagent of the assay). Data pointsin Quadrant 2 (Q2) in the graphs corresponds to cells that are positivefor the apoptosis detection reagent and positive for the necrosisdetection reagent of the assay (indicative of late-stage apoptosis).

FIGS. 5A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Huh7 cells. FIG. 5A shows the results forno-drug control. FIG. 5B shows the results for treatment with 10 μMSK1-I. FIG. 5C shows the results for treatment with 20 μM SK1-I. FIG. 5Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Huh7 cells.

FIGS. 6A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for PLC-PRF5 cells. FIG. 6A shows the resultsfor no-drug control. FIG. 6B shows the results for treatment with 10 μMSK1-I. FIG. 6C shows the results for treatment with 20 μM SK1-I. FIG. 6Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the PLC-PRF5 cells.

FIGS. 7A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Hep 3B cells. FIG. 7A shows the resultsfor no-drug control. FIG. 7B shows the results for treatment with 10 μMSK1-I. FIG. 7C shows the results for treatment with 20 μM SK1-I. FIG. 7Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Hep 3B cells.

FIGS. 8A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Hep G2 cells. FIG. 8A shows the resultsfor no-drug control. FIG. 8B shows the results for treatment with 10 μMSK1-I. FIG. 8C shows the results for treatment with 20 μM SK1-I. FIG. 8Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Hep G2 cells.

FIGS. 9A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Huh7 cells. FIG. 9A shows the resultsfor no-drug control. FIG. 9B shows the results for treatment with 5 μMozanimod. FIG. 9C shows the results for treatment with 10 μM ozanimod.FIG. 9D shows the results for treatment with 20 μM ozanimod. Ozanimodstrongly induced apoptosis in the Huh7 cells.

FIGS. 10A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for PLC-PRF5 cells. FIG. 10A shows theresults for no-drug control. FIG. 10B shows the results for treatmentwith 5 μM ozanimod. FIG. 10C shows the results for treatment with 10 μMozanimod. FIG. 10D shows the results for treatment with 20 μM ozanimod.Ozanimod strongly induced apoptosis in the PLC-PRF5 cells.

FIGS. 11A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep 3B cells. FIG. 11A shows theresults for no-drug control. FIG. 11B shows the results for treatmentwith 10 μM ozanimod. FIG. 11C shows the results for treatment with 20 μMozanimod. FIG. 11D shows the results for treatment with 40 μM ozanimod.Ozanimod strongly induced apoptosis in the Hep 3B cells.

FIGS. 12A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep G2 cells. FIG. 12A shows theresults for no-drug control. FIG. 12B shows the results for treatmentwith 10 μM ozanimod. FIG. 12C shows the results for treatment with 20 μMozanimod. FIG. 12D shows the results for treatment with 40 μM ozanimod.Ozanimod strongly induced apoptosis in the Hep G2 cells.

FIGS. 13A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Huh7 cells. FIG. 13A shows the resultsfor no-drug control. FIG. 13B shows the results for treatment with 10 μMABC294640. FIG. 13C shows the results for treatment with 20 μMABC294640. FIG. 13D shows the results for treatment with 40 μMABC294640. ABC294640 failed to induce apoptosis in the Huh7 cells.

FIGS. 14A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for PLC-PRF5 cells. FIG. 14A shows theresults for no-drug control. FIG. 14B shows the results for treatmentwith 10 μM ABC294640. FIG. 14C shows the results for treatment with 20μM ABC294640. FIG. 14D shows the results for treatment with 40 μMABC294640. ABC294640 did not substantially induce apoptosis in thePLC-PRF5 cells.

FIGS. 15A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep 3B cells. FIG. 15A shows theresults for no-drug control. FIG. 15B shows the results for treatmentwith 10 μM ABC294640. FIG. 15C shows the results for treatment with 20μM ABC294640. FIG. 15D shows the results for treatment with 40 μMABC294640. ABC294640 did not substantially induce apoptosis in the Hep3B cells.

FIGS. 16A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep G2 cells. FIG. 16A shows theresults for no-drug control. FIG. 16B shows the results for treatmentwith 10 μM ABC294640. FIG. 16C shows the results for treatment with 20μM ABC294640. FIG. 16D shows the results for treatment with 40 μMABC294640. ABC294640 failed to induce apoptosis in the Hep G2 cells.

FIGS. 17A-C show apoptosis assay data for various concentrations ofSK1-I and no-drug control for Jurkat cells (human T-cell leukemia cellline). FIG. 17A shows the results for no-drug control. FIG. 17B showsthe results for treatment with 10 μM SK1-I. FIG. 17C shows the resultsfor treatment with 20 μM SK1-I. SK1-I strongly induced apoptosis in theJurkat cells.

FIGS. 18A-C show apoptosis assay data for various concentrations ofozanimod and no-drug control for Jurkat cells. FIG. 18A shows theresults for no-drug control. FIG. 18B shows the results for treatmentwith 5 μM ozanimod. FIG. 18C shows the results for treatment with 10 μMozanimod. Ozanimod strongly induced apoptosis in the Jurkat cells.

In still further experiments, the effects of SK1-I and ozanimod onnormal (non-cancerous) primary human hepatocytes were investigated.Fresh human hepatocytes in a 12-well plate (HUF12) were obtained fromTriangle Research Labs (Durham, N.C., USA; part of Lonza Group) andhandled according to the supplier's protocol. The shipping medium wasaspirated from each well and replaced with 1 ml per well of warmHepatocyte Maintenance Medium. The plate was then placed in a 5% CO₂incubator at 37° C. and the hepatocytes were allowed to acclimateovernight. The Hepatocyte Maintenance Medium was replaced beforetreatment with drug or no-drug control, and the hepatocytes were treatedwith 0 μM (no-drug control), 10 μM, 20 μM, or 40 μM SK1-I or 0 μM, 2.5μM, 5 μM, or 10 μM ozanimod for 24 hours under incubation. The cellswere then harvested and analyzed using the GFP Certified®Apoptosis/Necrosis detection kit. The results are shown in FIGS. 19A-Dfor SK1-I and FIGS. 20A-D for ozanimod as follows.

FIGS. 19A-D show apoptosis assay data for various concentrations ofSK1-I and no-drug control for primary human hepatocytes. FIG. 19A showsthe results for no-drug control. FIG. 19B shows the results fortreatment with 10 μM SK1-I. FIG. 19C shows the results for treatmentwith 20 μM SK1-I. FIG. 19D shows the results for treatment with 40 μMSK1-I. SK1-I did not induce apoptosis of the primary human hepatocytesat any concentration tested.

FIGS. 20A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for primary human hepatocytes. FIG. 20Ashows the results for no-drug control. FIG. 20B shows the results fortreatment with 2.5 μM ozanimod. FIG. 20C shows the results for treatmentwith 5 μM ozanimod. FIG. 20D shows the results for treatment with 10 μMozanimod. Ozanimod did not induce apoptosis of the primary humanhepatocytes at any concentration tested.

Without limitation, the following embodiments are also provided.

Embodiments Involving Sphingosine Kinase 1 (SphK1) Inhibitors Embodiment1

A method for treating liver cancer in a mammalian subject, such as ahuman, including the step of:

-   -   administering to a mammalian subject in need of treatment for        liver cancer, an effective amount of a sphingosine kinase type I        inhibitor.

Embodiment 2

The method of embodiment 1, wherein the liver cancer is hepatic cellcarcinoma (HCC).

Embodiment 3

The pharmaceutical composition of embodiment 1, wherein the liver canceris selected from the group consisting of fibrolamellar HCC,cholangiocarcinoma (bile duct cancer) and angiosarcoma.

Embodiment 4

The method of any one of the preceding embodiments, wherein thesphingosine kinase type I inhibitor at least substantially does notinhibit sphingosine kinase type II.

Embodiment 5

The method of any one of the preceding embodiments, wherein thesphingosine kinase type I inhibitor includes a sphingosine kinase type Iinhibitor disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, or apharmaceutically acceptable salt thereof.

Embodiment 6

The method of any one of embodiments 1-5, wherein the sphingosine kinasetype I inhibitor includes SK1-I or a pharmaceutically acceptable saltthereof.

Embodiment 7

The method of any one of the preceding embodiments, wherein saidadministration includes parenteral administration.

Embodiment 8

The method of embodiment 7, wherein said administration is viainjection, such as intravenous injection, intramuscular injection, orsubcutaneous injection.

Embodiment 9

The method of any one of embodiments 1-6, wherein said administrationincludes non-parenteral administration.

Embodiment 10

The method of any one of embodiments 1-6, wherein said administrationincludes oral administration by ingestion.

Embodiment 11

The method of embodiment 10, wherein said oral administration includesadministering a dosage form including the sphingosine kinase type Iinhibitor and at least one pharmaceutically acceptable excipient.

Embodiment 12

The method of embodiment 11, wherein the dosage form is selected fromthe group consisting of a tablet, a capsule, and a gel cap.

Embodiment 13

The method of any one of embodiments 1-6, wherein said administrationincludes administration via the alimentary canal.

Embodiment 14

The method of any one of the preceding embodiments, further includingthe step of:

-   -   co-administering to the subject an effective amount of ozanimod        or a pharmaceutically acceptable salt thereof.

Embodiment 15

A pharmaceutical composition for the treatment of a liver cancer in amammalian subject, such as a human, including:

-   -   a therapeutically effective amount of a sphingosine kinase type        I inhibitor.

Embodiment 16

The pharmaceutical composition of embodiment 15, wherein the livercancer is hepatic cell carcinoma (HCC).

Embodiment 17

The pharmaceutical composition of embodiment 15, wherein the livercancer is selected from the group consisting of Fibrolamellar HCC,Cholangiocarcinoma (bile duct cancer) and Angiosarcoma.

Embodiment 18

The pharmaceutical composition of any one of the preceding embodiments,wherein the sphingosine kinase type I inhibitor at least substantiallydoes not inhibit sphingosine kinase type II.

Embodiment 19

The pharmaceutical composition of any one of the preceding embodiments,wherein the sphingosine kinase type I inhibitor includes a sphingosinekinase type I inhibitor disclosed in U.S. Pat. Nos. 8,372,888 and/or8,314,151, or a pharmaceutically acceptable salt thereof.

Embodiment 20

The pharmaceutical composition of any one of embodiment 15-18, whereinthe sphingosine kinase type I inhibitor includes SK1-I or apharmaceutically acceptable salt thereof.

Embodiment 21

The pharmaceutical composition of any one of the preceding embodiments,wherein said composition is for parenteral administration.

Embodiment 22

The pharmaceutical composition of embodiment 21, wherein saidcomposition is for administration via injection, such as intravenousinjection, intramuscular injection, or subcutaneous injection.

Embodiment 23

The pharmaceutical composition of any one of embodiments 15-20, whereinsaid composition is for non-parenteral administration.

Embodiment 24

The pharmaceutical composition of any one of embodiments 15-20, whereinsaid composition is for oral administration by ingestion.

Embodiment 25

The pharmaceutical composition of any one of embodiment 15-24, furtherincluding at least one pharmaceutically acceptable excipient.

Embodiment 26

The pharmaceutical composition of any one of embodiments 15-25, whereinsaid composition is a solid dosage form.

Embodiment 27

The pharmaceutical composition of embodiment 24, provided in a dosageform selected from the group consisting of a liquid, a tablet, acapsule, and a gel cap.

Embodiment 28

The pharmaceutical composition of any one of embodiments 15-20, whereinsaid composition is for administration via the alimentary canal.

Embodiment 29

The pharmaceutical composition of any one of embodiments 15-28, furtherincluding a therapeutically effective amount of ozanimod or apharmaceutically acceptable salt thereof.

Embodiment 30

A method for inducing apoptosis of mammalian liver cancer cells, such ashepatocellular carcinoma (HCC) cells, including the step of:

-   -   contacting the mammalian liver cancer cells with an effective        amount of a selective sphingosine kinase type I inhibitor, such        as any of those disclosed in U.S. Pat. Nos. 8,372,888 and/or        8,314,151, such as SK1-I, or a pharmaceutically acceptable salt        thereof.

Embodiment 31

Use of a selective sphingosine kinase type I inhibitor, such as any ofthose disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, such asSK1-I, or a pharmaceutically acceptable salt thereof, for inducingapoptosis of mammalian liver cancer cells, such as hepatocellularcarcinoma (HCC) cells.

Embodiments Involving Sphingosine-1-Phosphate Receptor AgonistsEmbodiment 32

A method for treating a cancer or a myleoproilferative disorder(myeloproliferative neoplasm) in a mammalian subject, such as a human,including the step of:

-   -   administering to a mammalian subject in need of treatment for a        cancer or myeloproliferative disorder, a therapeutically        effective amount of a sphingosine-1-phosphate receptor agonist,        such as an agonist of one or both of sphingosine-1-phosphate        receptor-1 (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅)        such as ozanimod (RPC1063) or a pharmaceutically acceptable salt        thereof.

Embodiment 33

The method of embodiment 32, wherein the myeloproliferative disorders isselected from the group consisting of: Chronic myelogenous leukemia(e.g, BCR-ABL1-positive); Chronic neutrophilic leukemia; Polycythemiavera; Primary myelofibrosis; Essential thrombocythemia; Chroniceosinophilic leukemia (not otherwise specified); and Mastocytosis.

Embodiment 34

The method of embodiment 32, wherein said cancer is a hematologicalmalignancy.

Embodiment 35

The method of embodiment 34, wherein said hematological malignancy isselected from the group consisting of: leukemias, lymphomas andmyelomas.

Embodiment 36

The method of embodiment 35, wherein said hematological malignancy isselected from the group consisting of: Acute lymphoblastic leukemia(ALL); Acute myelogenous leukemia (AML); Chronic lymphocytic leukemia(CLL); Chronic myelogenous leukemia (CML); Acute monocytic leukemia(AMoL); Hodgkin's lymphomas (e.g., any of main four subtypes); andNon-Hodgkin's lymphomas (any subtype).

Embodiment 37

The method of embodiment of embodiment 32, wherein said cancer is asolid organ cancer.

Embodiment 38

The method of embodiment 32, wherein the solid organ cancer is selectedfrom the group consisting of: Adipose tissue cancers such asLiposarcoma, Myxoid liposarcoma adipose; Bladder cancer; Bone cancerssuch as Chondroblastoma, Chordoma, Ewings sarcoma, Osteosarcoma, Spindlecell tumor; Brain tumors such as Ganglioneuroblastoma, Ganglioneuroma,Glioblastoma, Malignant peripheral nerve sheath tumor, Neuroblastoma,Neurofibroma, Schwannoma brain; Connective tissue cancers such asChondromyxoid fibroma, Chondrosarcoma, Dedifferentiated chondrosarcoma,Fibromatosis, Monophasic synovial sarcoma; Esophageal adenocarcinoma;Oral squamous cell carcinoma; Kidney cancers such as Kidney carcinoma,Renal cell carcinoma; Liver cancers such as Hepatocellular carcinoma(HCC), Fibrolamellar HCC, Cholangiocarcinoma (bile duct cancer) andAngiosarcoma; Lung cancer such as NSCLC, SCLC; Uterine tumors; Head andNeck cancers such as head and neck squamous cell carcinoma; Ovariantumors; Prostate cancer; Muscle tissue cancers such as Acutequadriplegic myopathy; Skin cancers such as Melanoma, Sarcoma, Kaposisarcoma; Alveolar rhabdomyo sarcoma, Embryonal rhabdomyo sarcoma,Leiomyosarcoma; Germ cell tumors such as of the testes, testicularcancer; and Thyroid cancer such as Thyroid adenocarcinoma.

Embodiment 39

The method of embodiment 33, wherein the solid organ cancer is livercancer.

Embodiment 40

The method of embodiment 39, wherein the liver cancer is hepatic cellcarcinoma (HCC).

Embodiment 41

The method of any one of embodiments 32-40, wherein thesphingosine-1-phosphate receptor agonist at least substantially does notagonize sphingosine-1-phosphate receptors other than types-1 and -5.

Embodiment 42

The method of any one of embodiments 32-41, wherein thesphingosine-1-phosphate receptor agonist includes asphingosine-1-phosphate receptor agonist disclosed in any of U.S. Pub.Nos. 20110172202, 20130231326, and 20150299149 or a pharmaceuticallyacceptable salt thereof.

Embodiment 43

The method of any one of embodiments 32-42, wherein thesphingosine-1-phosphate receptor agonist includes ozanimod or apharmaceutically acceptable salt thereof.

Embodiment 44

The method of any one of embodiments 32-43, wherein said administrationincludes parenteral administration.

Embodiment 45

The method of embodiment 44, wherein said administration is viainjection, such as intravenous injection, intramuscular injection, orsubcutaneous injection.

Embodiment 46

The method of any one of embodiments 32-43, wherein said administrationincludes non-parenteral administration.

Embodiment 47

The method of any one of embodiments 32-43, wherein said administrationincludes oral administration by ingestion.

Embodiment 48

The method of embodiment 47, wherein said oral administration includesadministering a dosage form including the sphingosine-1-phosphatereceptor agonist and at least one pharmaceutically acceptable excipient.

Embodiment 49

The method of embodiment 48, wherein the dosage form is selected fromthe group consisting of a liquid, a tablet, a capsule, and a gel cap.

Embodiment 50

The method of any one of embodiments 32-43, wherein said administrationincludes administration via the alimentary canal.

Embodiment 51

The method of any one of embodiments 32-50, further including the stepof:

-   -   co-administering to the subject an effective amount of a        sphingosine kinase type I inhibitor, such as SK1-I, or a        pharmaceutically acceptable salt thereof.

Embodiment 52

The method of any one of embodiments 32-51, further including the stepof:

-   -   co-administering to the subject a therapeutically effective        amount of a cellular ceramide generation promoter, such as        6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic        acid]cyclosporin D (Valspodor; PSC833) or a pharmaceutically        acceptable salt thereof.

Embodiment 53

The method of any one of embodiments 32-52, further including the stepof:

-   -   coadministering to the subject a therapeutically effective        amount of ceramide.

Embodiment 54

A pharmaceutical composition for the treatment of a cancer or amyeloproliferative disorder (myeloproliferative neoplasm) in a mammaliansubject, such as a human, including:

-   -   a therapeutically effective amount of a sphingosine-1-phosphate        receptor agonist, such as an agonist of one or both of        sphingosine-1-phosphate receptor-1 (S1P₁) and        sphingosine-1-phosphate receptor-5 (S1P₅) such as ozanimod        (RPC1063) or a pharmaceutically acceptable salt thereof.

Embodiment 55

The pharmaceutical composition of embodiment 54, wherein themyeloproliferative disorders is selected from the group consisting of:Chronic myelogenous leukemia (BCR-ABL1-positive); Chronic neutrophilicleukemia; Polycythemia vera; Primary myelofibrosis; Essentialthrombocythemia; Chronic eosinophilic leukemia (not otherwisespecified); and Mastocytosis.

Embodiment 56

The pharmaceutical composition of embodiment 54, wherein said cancer isa hematological malignancy.

Embodiment 57

The pharmaceutical composition of embodiment 56, wherein saidhematological malignancy is selected from the group consisting of:leukemias, lymphomas and myelomas.

Embodiment 58

The pharmaceutical composition of embodiment 57, wherein saidhematological malignancy is selected from the group consisting of: Acutelymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); Chroniclymphocytic leukemia (CLL); Chronic myelogenous leukemia (CML); Acutemonocytic leukemia (AMoL); Hodgkin's lymphomas (e.g., any of main foursubtypes); and Non-Hodgkin's lymphomas (any subtype).

Embodiment 59

The pharmaceutical composition of embodiment of embodiment 54, whereinsaid cancer is a solid organ cancer.

Embodiment 60

The pharmaceutical composition of embodiment 59, wherein the solid organcancer is selected from the group consisting of: Adipose tissue cancerssuch as Liposarcoma, Myxoid liposarcoma adipose; Bladder cancer; Bonecancers such as Chondroblastoma, Chordoma, Ewings sarcoma, Osteosarcoma,Spindle cell tumor; Brain tumors such as Ganglioneuroblastoma,Ganglioneuroma, Malignant peripheral nerve sheath tumor, Neuroblastoma,Neurofibroma, Schwannoma brain; Connective tissue cancers such asChondromyxoid fibroma, Chondrosarcoma, Dedifferentiated chondrosarcoma,Fibromatosis, Monophasic synovial sarcoma; Esophageal adenocarcinoma;Oral squamous cell carcinoma; Kidney cancers such as Kidney carcinoma,Renal cell carcinoma; Liver cancers such as Hepatocellular carcinoma(HCC), Fibrolamellar HCC, Cholangiocarcinoma (bile duct cancer) andAngiosarcoma; Lung cancer such as NSCLC, SCLC; Uterine tumors; Head andNeck cancers such as head and neck squamous cell carcinoma; Ovariantumors; Prostate cancer; Muscle tissue cancers such as Acutequadriplegic myopathy; Skin cancers such as Melanoma, Sarcoma, Kaposisarcoma; Alveolar rhabdomyo sarcoma, Embryonal rhabdomyo sarcoma,Leiomyosarcoma; Germ cell tumors such as of the testes, testicularcancer; and Thyroid cancer such as Thyroid adenocarcinoma.

Embodiment 61

The pharmaceutical composition of embodiment 59, wherein the solid organcancer is a liver cancer.

Embodiment 62

The pharmaceutical composition of embodiment 61, wherein the livercancer is selected from the group consisting of hepatic cell carcinoma(HCC), fibrolamellar HCC, cholangiocarcinoma (bile duct cancer) andangiosarcoma.

Embodiment 63

The pharmaceutical composition of any one of embodiments 54-62, whereinthe sphingosine-1-phosphate receptor agonist at least substantially doesnot agonize sphingosine-1-phosphate receptors other than types-1 and -5.

Embodiment 65

The pharmaceutical composition of any one of embodiments 54-64, whereinthe sphingosine-1-phosphate receptor agonist includes asphingosine-1-phosphate receptor agonist disclosed in any of U.S. PubNos. 20110172202, 20130231326, and 20150299149.

Embodiment 66

The pharmaceutical composition of any one of embodiments 54-64, whereinthe sphingosine-1-phosphate receptor agonist includes ozanimod or apharmaceutically acceptable salt thereof.

Embodiment 67

The pharmaceutical composition of any one of embodiments 54-66, whereinsaid composition is for parenteral administration.

Embodiment 68

The pharmaceutical composition of embodiment 67, wherein saidcomposition is for administration via injection, such as intravenousinjection, intramuscular injection, or subcutaneous injection.

Embodiment 69

The pharmaceutical composition of any one of embodiments 54-66, whereinsaid composition is for non-parenteral administration.

Embodiment 70

The pharmaceutical composition of any one of embodiments 54-66, whereinsaid composition is for oral administration by ingestion.

Embodiment 71

The pharmaceutical composition of any one of embodiments 54-70, furtherincluding at least one pharmaceutically acceptable excipient.

Embodiment 72

The pharmaceutical composition of any one of embodiments 54-71, whereinsaid composition is a solid dosage form.

Embodiment 73

The pharmaceutical composition of embodiment 72, provided in a dosageform selected from the group consisting of a tablet, a capsule, and agel cap.

Embodiment 74

The pharmaceutical composition of any one of embodiments 54-66, whereinsaid composition is for administration via the alimentary canal.

Embodiment 75

The pharmaceutical composition of any one of embodiments 54-74, furtherincluding a therapeutically effective amount of a sphingosine kinasetype I inhibitor, such as one disclosed in U.S. Pat. Nos. 8,372,888and/or 8,314,151, such as SK1-I, or a pharmaceutically acceptable saltthereof.

Embodiment 76

The pharmaceutical composition of any one of embodiments 54-74, for usein combination with a therapeutically effective amount of a sphingosinekinase type I inhibitor, such as one disclosed in U.S. Pat. Nos.8,372,888 and/or 8,314,151, such as SK1-I, or a pharmaceuticallyacceptable salt thereof.

Embodiment 77

The pharmaceutical composition of any one of embodiments 54-76, furtherincluding a therapeutically effective amount of a cellular ceramidegeneration promoter, such as6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporinD (Valspodor; PSC833) or a pharmaceutically acceptable salt thereof.

Embodiment 78

The pharmaceutical composition of any one of embodiments 54-76, for usein combination with a therapeutically effective amount of a cellularceramide generation promoter,6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporinD (Valspodor; PSC833) or a pharmaceutically acceptable salt thereof.

Embodiment 79

The pharmaceutical composition of any one of embodiments 54-78 for usein combination with a therapeutically effective amount of ceramide.

Embodiment 80

A method for inducing apoptosis of mammalian cancer cells, such as livercancer cells, such as hepatocellular carcinoma (HCC) cells, includingthe step of:

-   -   contacting the mammalian cancer cells with an effective amount        of a sphingosine-1-phosphate receptor agonist, such as an        agonist of one or both of sphingosine-1-phosphate receptor-1        (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅) such as any        of those disclosed in U.S. Pub Nos. 20110172202, 20130231326,        and 20150299149, such as ozanimod (RPC1063), or a        pharmaceutically acceptable salt thereof.

Embodiment 81

Use of a sphingosine-1-phosphate receptor agonist, such as an agonist ofone or both of sphingosine-1-phosphate receptor-1 (S1P₁) andsphingosine-1-phosphate receptor-5 (S1P₅) such as any of those disclosedin U.S. Pub Nos. 20110172202, and 20130231326, and 20150299149, such asozanimod (RPC1063), or a pharmaceutically acceptable salt thereof, forinducing apoptosis of mammalian cancer cells, such as liver cancercells, such as, hepatocellular carcinoma (HCC) cells.

For embodiments involving ceramide, the ceramide may, for example, beformulated/co-formulated in an acid stable lipid vesicle/particlecomposition as disclosed in U.S. Pub. No. 20140271824, which is herebyincorporated by reference in its entirety, andadministered/co-administered, for example by injection, such asintravenous injection, or orally.

Any of the method of treatment/use embodiments set forth herein mayfurther include the step of: co-administering one or more immunecheckpoint inhibitors, which may be monoclonal antibodies (mABs). Theimmune checkpoint inhibitor may be selected from the group consisting ofthe following: PD-1 inhibitors such as mAbs Pembrolizumab (Keytruda®)and Nivolumab (Opdivo®); PD-L1 inhibitors such as mAbs Atezolizumab(Tecentriq®), Avelumab (Bavencio®), and Durvalumab (Imfinzi®); andCTLA-4 inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain IgSuppressor of T Cell Activation (VISTA) inhibitors such as mAbJNJ-61610588 (ImmuNext Inc.). Similarly, any of the pharmaceuticalcomposition embodiments of the invention may be for use in combinationwith one or more immune checkpoint inhibitors, such as those disclosedherein.

Still another embodiment of the invention provides a method for treatinga cancer or a myleoproliferative disorder (myeloproliferative neoplasm),such as any of those disclosed herein, for example, a liver cancer, in amammalian subject, such as a human, including the step of:

-   -   co-administering to a mammalian subject in need of treatment for        a cancer or myeloproliferative disorder, a therapeutically        effective amount of:        -   (a) a sphingosine kinase type I inhibitor, such as one            disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, such            as SK1-I, or a pharmaceutically acceptable salt thereof; and        -   (b) one or more immune checkpoint inhibitors, which may be            monoclonal antibodies, such as one or more selected from the            group consisting of: PD-1 inhibitors such as mAbs            Pembrolizumab (Keytruda®) and Nivolumab (Opdivo®); PD-L1            inhibitors such as mAbs Atezolizumab (Tecentriq®), Avelumab            (Bavencio®), and Durvalumab (Imfinzi®); and CTLA-4            inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain Ig            Suppressor of T Cell Activation (VISTA) inhibitors such as            mAb JNJ-61610588 (ImmuNext Inc.).

Immune checkpoint inhibitors may, for example, be administered byinjection in the dosages described herein and/or at the currentlyapproved dosages for said inhibitors.

The amount of compound that is effective for the treatment or preventionof a condition, alone or in combination with other compounds, may bedetermined by standard techniques. In addition, in vitro and/or in vivoassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed will also depend on, e.g., theroute of administration and the seriousness of the condition, and can bedecided according to the judgment of a practitioner and/or eachpatient's circumstances. In other examples thereof, variations willnecessarily occur depending upon the weight and physical condition(e.g., hepatic and renal function) of the patient being treated, theaffliction to be treated, the severity of the symptoms, the frequency ofthe dosage interval, the presence of any deleterious side-effects, andthe particular compound utilized, among other things.

Administration may be as a single dose or as a divided dose. In oneembodiment, an effective dosage is administered once per month until thecondition is abated. In another embodiment, the effective dosage isadministered once per week, or twice per week or three times per weekuntil the condition is abated. An effective dosage may, for example, beadministered at least once daily or at least or at least once everytwo-days, or at least once every three days, four days, five days, sixdays or seven days. In another embodiment, an effective dosage amount isadministered about every 24 h until the condition is abated. In anotherembodiment, an effective dosage amount is administered about every 12 huntil the condition is abated. In another embodiment, an effectivedosage amount is administered about every 8 h until the condition isabated. In another embodiment, an effective dosage amount isadministered about every 6 h until the condition is abated. In anotherembodiment, an effective dosage amount is administered about every 4 huntil the condition is abated.

The therapeutically effective dose may be expressed in terms of theamount of the compound(s) or pharmaceutically acceptable salts thereofadministered per unit body weight of the subject per day of treatment,or the total amount administered per day of treatment. A daily dose may,for example, be at least 0.005 mg/kg of body weight, at least 0.01 mg/kgof body weight, at least 0.025 mg/kg of body weight, at least 0.05 mg/kgof body weight, at least 0.1 mg/kg of body weight, at least 0.2 mg/kg ofbody weight, at least 0.3 mg/kg of body weight, at least 0.4 mg/kg ofbody weight, at least 0.5 mg/kg of body weight, at least 0.6 mg/kg ofbody weight, at least 0.7 mg/kg of body weight, at least 0.8 mg/kg ofbody weight, at least 0.9 mg/kg of body weight, at least 1 mg/kg of bodyweight, at least 1.5 mg/kg of body weight, at least 2 mg/kg of bodyweight, at least 2.5 mg/kg of body weight, at least 3 mg/kg of bodyweight, at least 3.5 mg/kg of body weight, at least 4 mg/kg of bodyweight, at least 4.5 mg/kg of body weight, at least 5 mg/kg of bodyweight, or at one of said doses. A total daily dose may, for example, bein the range of 0.005 mg/kg to 5 mg/kg or any subrange or value therein,such as 0.025 to 5 mg/kg body weight, such as 0.05 to 5 mg/kg bodyweight. A total daily dose may, for example be in the range of 0.1 mg to1,000 mg total or any subrange or value therein, such as 0.1 mg to 1,000mg, such as 0.1 mg to 100 mg, such as 0.1 mg to 50 mg, such as 0.5 mg to50 mg, such as 1.0 mg to 50 mg, such as 5 mg to 50 mg, or 0.1 mg to 10mg, such as 0.5 mg to 10 mg. For SK1-I and related SphK1 inhibitorsdisclosed U.S. Pat. Nos. 8,372,888 and 8,314,151, and pharmaceuticallyacceptable salts thereof, a daily dose for human subjects may, forexample, also be in the range of 0.5 mg/kg to 5 mg/kg or any subrange orvalue therein, such as 1 mg/kg to 4 mg/kg, such as 1 mg/kg to 3 mg/kg,or, for example, a total daily dose of 5 mg to 50 mg or any subrange orvalue therein, such as 10 mg to 40 mg, such as 20 mg to 40 mg. Forozanimod and related sphingosine-1-phosphate receptor agonists disclosedin U.S. Pub Nos. 20110172202, 20130231326, and 20150299149, andpharmaceutically acceptable salts thereof, a daily dose for humansubjects may, for example, also be in the range of 0.1 mg to 10 mg orany subrange or value therein, such as 0.1 mg to 5 mg, such as 0.5 to 5mg, such as 0.5 mg to 2.5 mg, such as 0.5 mg to 1.5 mg. A pharmaceuticalcomposition according to the invention may, for example, include a dailydose amount of the compound as set forth herein.

The duration of treatment by administration of a therapeutic compound orcombination according to the invention may continue for a plurality ofdays, such as for at least one week, at least two weeks, at least threeweeks, at least four weeks, at least two months, at least three months,at least four months, at least five months, at least six months, atleast seven months, at least eight months, at least nine months, atleast 10 months, at least 11 months, at least 12 months, at least 1½years, at least 2 years, at least three years, at least four years, ormay continue indefinitely.

The terms co-administration and co-administering mean that each of thethings being co-administered is administered to a subject in suchtemporal proximity that each (or its active metabolite(s)) is present inactive form in the subject for an at least partially overlapping periodof time. Accordingly, co-administration may include, simultaneousadministration, such as when the things being administered are part ofthe same pharmaceutical composition, or sequential administration of thethings being co-administered, for example, within the same day of eachother, within 12 hours of each other, within 6 hours of each other,within 3 hours of each other, within 1 hours of each other, or within 15minutes of each other. The things being administered may be administeredby the same route, such as by oral ingestion or injection, or bydifferent routes.

Pharmaceutically acceptable salts and the selection and preparationthereof are well known in the art. Such salts include but are notlimited to hydrochloride, citrate, glycolate, fumarate, malate,tartrate, mesylate, esylate, cinnamate, isethionate, sulfate, phosphate,diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate,acetate, dichloroacetate, lactate, p-toluenesulfonate, pamitate,pidolate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamidobenzoate, glutamate, aspartate, glycolate, adipate, alginate, ascorbate,besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate,cyclamate, laurylsulfate, edisylate, gentisate, galactarate, gluceptate,gluconate, glucuronate, oxoglutarate, hippurate, lactobionate, malonate,maleate, mandalate, napsylate, napadisylate, oxalate, oleate, sebacate,stearate, succinate, thiocyanate, undecylenate, and xinafoate.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application to mean oneor more unless the context clearly dictates otherwise. Further, the term“or” is used in the present application to mean the disjunctive “or” orthe conjunctive “and.” It should also be understood that wherever in thepresent application the term comprising or including (or a term ofsimilar scope) is recited in connection with the description of anyembodiment or part thereof, a corresponding embodiment or part thereofreciting instead the term consisting essentially of or the termconsisting of (or a term of similar scope) is also disclosed.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s). Moreover,features described in connection with one embodiment of the inventionmay be used in conjunction with other embodiments, even if notexplicitly exemplified in combination within.

What is claimed is:
 1. A method for treating hepatocellular carcinoma(HCC) cancer in a mammalian subject, comprising the step of:administering to a mammalian subject in need of treatment forhepatocellular carcinoma, an effective amount of the compound

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the mammalian subject is a human.
 3. The method of claim 1,wherein the administering step comprises administering the hydrochloridesalt of the compound to the mammalian subject.
 4. The method of claim 3,wherein the mammalian subject is a human.
 5. The method of claim 1,further comprising the step of: co-administering to the mammaliansubject an effective amount of ozanimod or a pharmaceutically acceptablesalt thereof.
 6. The method of claim 5, wherein the co-administeringstep comprises co-administering to the mammalian subject thehydrochloride salt of ozanimod.
 7. The method of claim 2, furthercomprising the step of: co-administering to the mammalian subject aneffective amount of ozanimod or a pharmaceutically acceptable saltthereof.
 8. The method of claim 7, wherein the co-administering stepcomprises co-administering to the mammalian subject the hydrochloridesalt of ozanimod.
 9. The method of claim 3, further comprising the stepof: co-administering to the mammalian subject an effective amount ofozanimod or a pharmaceutically acceptable salt thereof.
 10. The methodof claim 9, wherein the co-administering step comprises co-administeringto the mammalian subject the hydrochloride salt of ozanimod.
 11. Themethod of claim 4, further comprising the step of: co-administering tothe mammalian subject an effective amount of ozanimod or apharmaceutically acceptable salt thereof.
 12. The method of claim 11,wherein the co-administering step comprises co-administering to themammalian subject the hydrochloride salt of ozanimod.
 13. A method forinducing apoptosis of mammalian hepatocellular carcinoma (HCC) cells,comprising the step of: contacting the mammalian hepatocellularcarcinoma cells with an effective amount of the compound

or a pharmaceutically acceptable salt thereof.
 14. The method of claim13, wherein the mammalian hepatocellular carcinoma cells are humanhepatocellular carcinoma cells.
 15. The method of claim 13, furthercomprising the step of: contacting the mammalian hepatocellularcarcinoma cells with an effective amount of ozanimod or apharmaceutically acceptable salt thereof.
 16. The method of claim 15,wherein the mammalian hepatocellular carcinoma cells are humanhepatocellular carcinoma cells.
 17. A pharmaceutical compositioncomprising: the compound

or a pharmaceutically acceptable salt thereof; and ozanimod or apharmaceutically acceptable salt thereof.
 18. The pharmaceuticalcomposition of claim 17, further comprising at least onepharmaceutically acceptable excipient.
 19. The pharmaceuticalcomposition of claim 17, comprising the hydrochloride salt of thecompound and the hydrochloride salt of ozanimod.
 20. The pharmaceuticalcomposition of claim 19, further comprising at least onepharmaceutically acceptable excipient.